The epidermis of the skin is a stratified squamous epithelium, which plays an important protective role. It manifests this role by building an extensive cytoskeletal architecture, the unique feature of which is the presence of keratin filaments. There are two major pairs of keratins in the epidermis: one pair is expressed in dividing cells and the other expressed in terminally differentiating cells (E Fuchs, Epidermal differentiation and keratin gene expression, J Cell Sci Suppl, 7:197-208; 1993).
For terminal differentiation, epidermal cells move from the basal layer through the spinous layer and the granular layer towards the stratum corneum. During this process, they develop from mitotically active cells into dead, flattened squames. At the various stages of this development, different proteins are expressed. Crosslinking of epidermal proteins eventually leads to the establishment of the cornified envelope, a thick peripheral protein envelope that stabilizes each corneocyte. Additionally, lipids are synthesized in lamellar granules which are subsequently extruded into the extracellular space where they surround the corneocytes and build the lipid envelope. The stratum corneum is an impermeable, insoluble, and highly protective fortress, which keeps microorganisms out and essential bodily fluids in.
The squamous stratified epithelium of the skin is one of the most important barriers of the body, separating it from the surrounding environment and preventing the loss of water and solutes. For decades, the barrier function has mainly been ascribed to the stratum corneum with its corneocytes, cornified envelope and intercellular lipid accumulations: the role of tight junctions (TJs) in the barrier function of epidermis has, in general, been overlooked. However, with recent findings, the role and importance of TJs has come to the forefront. TJs are very complex structures that are formed by transmembrane, plaque and scaffolding proteins. Transmembrane proteins, that is, the family of claudins, occludins and the family of junctional adhesion molecules (JAMs), and scaffolding proteins, such as the zonula occludens (ZO), are important for the formation and regulation of the permeability barrier and for the formation of a molecular fence that separates lipids from apical and basolateral parts of the cell; are contact sites for cell surface receptors, for example, TGF-β-receptor, and molecules of signal transduction pathways; and are involved in the interaction with cells of the immune system, for example, neutrophils. They are often targets for pathogens and their toxins as well as allergens
Tight junctions are a dynamic structure with a plurality of distinct, yet somewhat interrelated, functions including permeability (barrier function), polarity (fence function), signaling (cell growth & differentiation), regulation (gene expression & cell proliferation), and tumor suppression. Of these, perhaps the most critical relative to skin aging and the maintenance of skin appearance is its permeability or barrier properties.
TJs form a diffusion barrier that regulates the flux of hydrophilic molecules through the paracellular pathway. Structurally, the TJs form a continuous network of parallel, interconnected intramembrane strands. The TJ strands consist of peripheral and integral membrane proteins that build up morphologically distinguishable strands and connect neighboring cells. Occludin was originally considered important in the formation and sealing of TJs, because the antibody for occludin recognized the strands of TJs. However, it was found that occludin was not essential in the formation of TJ strands, suggesting that it is a regulatory or more of a regulatory protein rather than a structural protein. Whereas only one occludin gene exists, claudin occurs in a multigene family with 24 or more forms. Claudin-1, -2 and -4 have been found to be essential for TJ function in functional analyses. Furthermore, studies of the expression of six different claudin proteins (claudin-1, -2, -3, -4, -5, and -7) in three tissues (liver, kidney, and pancreas) of aging male and female mice and found an age-dependent decrease in the expression of several claudin proteins in all three tissues observed. (T D'Souza, C A Sherman-Baust, S Poosala, J M Mullin and P J Morin, Age-Related Changes of Claudin Expression in Mouse Liver, Kidney, and Pancreas, The Journals of Gerontology Series A: Biological Sciences and Medical Sciences Advance Access published online on Aug. 19, 2009). This suggests another basis for the decrease in tissue barrier function and hence loss of hydration and the manifestation of skin aging in older individuals.
Another structure associated with the TJs is the desmosomes. Desmosomes are adhesive intercellular junctions that attach cell surface adhesion proteins to intracellular keratin filaments (E Delva et al, The Desmosomes, Cold Spring Harbor Perspective in Biology, 2009, 1:a002543). Abnormality in the desmosome-keratin filament complex leads to a breakdown in cell adhesion (fragility) and increase in Trans Epidermal Water Loss (TEWL). Key genes/proteins involved in maintaining desmosome functions include:                Desmogleins & Desmocollins (members of the Cadherin super family) which mediate adhesion at desmosomes; provide structural integrity of the epidermis; and modulate keratinocyte proliferation and differentiation;        Plakoglobin & Plakophillins which recruit intermediate filaments to sites of desmosome assembly and maintain desmosomal integrity        Desmoplakin which mediate linkage to the cytoskeleton-pivotal in the development of epidermis; and        Cadherin which mediates Ca2+-dependent contact between adjacent cells and whose lack of expression causes separation of keratinocytes & weakened desmosomal adhesion;        
Water homeostasis of the epidermis is essential for the normal function of the skin and for normal stratum corneum (SC) hydration. It is a determinant of skin appearance, mechanical properties, barrier function, and metabolism. In addition, it is indispensable to maintaining proper water balance of the body. Dehydration of SC is a typical feature of skin aging, especially in photo-aged skin, and of several diseases, for example, eczema, atopic dermatitis, psoriasis, and hereditary ichthyosis. (M Takenouchi, H Suzuki, H Tagami, Hydration characteristics of pathologic stratus corneum-evaluation of bound water, J Invest Dermatol, 87 (5):574-576, 1986; P Thune, Evaluation of the hydration and the water-holding capacity in atopic skin and so-called dry skin, Acta Derm Venerol Suppl, 144:133-135, 1989; R J Scheuplein, I H Blank, Permeability of the Skin, Physiol Rev, 51 (4):702-747, 1971). SC hydration also appears to be directly linked to hyperplasia and inflammation which argues for a biosensor function of water content (Y Ashida, M Ogo, M Denda, Epidermal interleukin-1-alpha generation is amplified at low humidity: implications for the pathogenesis of inflammatory dermatoses Br J Dermatol, 144 (2):238-243, 2001; M Denda, J Sato, T. Tsuchiya, Low humidity stimulates DNA synthesis and amplifies the hyperproliferative dermatoses, J Invest Dermatol, 111 (5): 873-878, 1998).
In order to maintain and improve skin hydration, one needs to address both water homeostasis and functions related to structural barrier mechanisms, such as, tight junction, desmosome and epidermal differentiation, thereby improving skin health and appearance.
Water homeostasis depends on several factors, for example, the supply of water from the body, water diffusion from the viable layers of the epidermis, trans-epidermal water loss (TEWL), and water-holding capacity of stratum corneum. Supply of water from the body depends on its water balance and putatively on blood circulation. Water diffusion through the epidermis depends on transcellular as well as paracellular pathways along osmotic gradients. Transcellular diffusion is performed through pores, i.e., proteins that act as pores, in the plasma membrane which are called aquaglyceroporins, a subgroup of the aquaporin (AQP) family, as well as directly through plasma membranes. Paracellular diffusion might be controlled by tight junctions (TJ) and TJ proteins. TEWL depends on the barrier function of the skin, which is influenced by, among others, TJ proteins; on the environmental conditions encountered, for example, high and low humidity and temperature; and on the water supply present. SC water-holding capacity is thought to depend on SC structure and composition, particularly the content of natural moisturizing factors and humectants like glycerol.
Aquaglyceroporins are best known and responsible for transporting both water and small neutral solutes, such as glycerol. One of the key aquaglyceroporins is water channel aquaporin 3 (AQP3). AQP3 is the most abundant AQP in human epidermis and is responsible for hydration in human skin epidermis. AQP3 was first cloned from rat kidney (S Sasaki, K Fushimi, H Saito, F Saito, S Uchida, K Ishibashi et al, Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct, J Clin Invest, 93:1250-1256, 1994) and subsequently found in red blood cells (N Roudier, J M Verbavatz, C Maurel, P Ripoche, F Tacnet, Evidence for the presence of aquaporin-3 in human red blood cells, J Biol Chem, 273:8407-8412, 1998), chondrocytes (A Mobasheri, E Trujillo, S Bell, S D Carter, P D Clegg, P Martin-Vasallo et al., Aquaporin water channels AQP1 and AQP3, are expressed in equine articular chondrocytes, Vet J, 168:143-150, 2004), and in epithelial cells from the urinary, digestive, and respiratory systems (A Frigeri, M A Gropper, F Umenishi, M Kawashima, D Brown, A Verkman A, Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues, J Cell Sci, 108:2993-3002, 1995). In skin, AQP3 is constitutively expressed by epidermal keratinocytes (R Sougrat, M Morand, C Gondran, P Barre, R Gobin, F Bonte et al., Functional expression of AQP3 in human skin epidermis and reconstructed epidermis, J Invest Dermatol, 118:678-685, 2002). AQP3-deficient mice suffer from reduced water and glycerol permeabilities and decreased water holding capacity of the stratum corneum, demonstrating a pivotal role of this channel in the maintenance of skin hydration (T Ma, M Hara, R Sougrat, J M Verbavatz, A S Verkman, Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3, J Biol Chem, 277:17147-17153, 2002). AQP3-deficient mice also show delayed barrier recovery after tape-stripping disruption and delayed wound healing (M Hara, T Ma, A S Verkman, Selectively reduced glycerol in skin of aquaporin-3-deficient mice may account for impaired skin hydration, elasticity, and barrier recovery, J Biol Chem, 277:46616-46621, 2002), suggesting a possible role of AQP3 in the regulation of keratinocyte differentiation and proliferation.
One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin (C Cao, S Wan, Q Jiang, A Amaral, S Lu, G Hu, Z Bi, N Kouttab, W Chu, Y Wan, J Cell Physiol, 215 (2):506-516, 2008). Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). It has been shown that UV induces aquaporin-3 (AQP3) down-regulation in human skin keratinocytes.
AQP3 may also play a role in sebaceous gland physiology, as it is expressed in the sebaceous gland (A Frigeri, M A Cropper, F Umenishi, M Kawashima, D Brown and A S Verkman, Localization of MIWC and CLIP water channel homologs in neuromuscular, epithelial and glandular tissues. J Cell Sci 108:2993-3002, 1995). Analysis of sebaceous gland-deficient mice suggested that sebaceous gland-derived glycerol is an important contributor to SC hydration (J W Fluhr, M Mao-Qiang, B E Brown, P W Wertz, D Crumrine, J P Sundberg, K R Feingold and P M Elias, Glycerol regulates stratum corneum hydration in sebaceous gland deficient (asebia) mice, J Invest Dermatol 120:728-737, 2003). Another group reported co-localization of AQP3 with phospholipase D2 in keratinocytes and suggested that phosphatidylglycerol synthesis might be facilitated by AQP3-mediated glycerol transport and phospholipase D2 action (X Zheng, and W B Bollag, Aquaporin 3 colocates with phospholipase D2 in caveolin-rich membrane microdomains and is down-regulated upon keratinocyte differentiation, J Invest Dermatol 121:1487-1495, 2003). Phospholipids including phosphatidylglycerol are involved in epidermal lipid biosynthesis, which are important in maintaining lamellar lipid structure and SC barrier function.
In summary, AQP3 has been shown to play an important role in epidermal glycerol transport and steady-state accumulation of glycerol in epidermis and SC providing a rational scientific basis for the longstanding practice of including glycerol in cosmetic and skin medicinal preparations. Thus, activation/up-regulation of AQP3 should improve skin hydration, barrier function and skin appearance, possibly reducing skin sagging and wrinkling.
Retinoids are important regulators of several biological processes such as embryogenesis, reproduction, differentiation, proliferation, and apoptosis. By regulating keratinocyte proliferation and differentiation, retinoids increase stratum granulosum thickness and are widely used in cosmetics for the treatment of skin aging. Retinoic acid has been shown recently to stimulate AQP3 gene and protein expression in normal human epidermal keratinocytes (NHEK) as well as in skin explants and to increase glycerol transport capacity, indicating that stimulation of AQP3 expression was accompanied by an enhancement of biological activity. Over expression of functional AQP3 may increase skin glycerol content, which in turn may be a key messenger of keratinocyte proliferation and early differentiation processes. The recent finding that retinoids increase AQP3 expression and stimulate glycerol transport further confirms that, beyond its humectant properties, glycerol may actually play a biological role in epidermal maturation.
However, there are contradictory viewpoints which suggest that over-expression of AQP3, at least in part, accounts for skin dryness. Phenotypical studies in AQP3 null mice indicate that AQP3 plays an important role in epidermal glycerol and water transport and that up-regulation of AQP3 leads to more water movement from dermis to epidermis. In AQP3-deficient mice, skin barrier recovery and wound healing is significantly delayed, while up-regulation of AQP3 facilitates epidermal cell migration during wound healing. However, hyper-expression of AQP3 is also associated with the increase of TEWL. A decrease in the water-holding capacity of the stratum corneum combined with an increase in water transport to the stratum corneum may lead to more water loss and skin dryness. This is consistent with the finding that increased AQP3 expression is found in the epidermis of patients with atopic eczema (AE), a diSease characterized by dry skin, in contrast with that of healthy skin. Patients with AE show defective skin barrier function and reduced water-holding capacity in stratum corneum which are believed to contribute to increased water loss and dry skin in AE. These findings demonstrate that increased expression of AQP3 may lead to increased water loss in AE. However, improving barrier function in AE may resolve excess water loss and dryness.
All-trans retinoic acid (atRA) has been shown to up-regulate the expression of AQP3. (G Bellemère, Von Stetten and T Oddos, Retinoic Acid Increases Aquaporin 3 Expression in Normal Human Skin, J Invest Dermatol, 128:542-548, 2008.) Nicotinamide has been shown to decrease the expression of AQP3 and water permeability induced by all-trans retinoic acid (atRA) in a concentration-dependent manner. Specifically, nicotinamide attenuates atRA-induced AQP3 hyper-expression. These finding may further explain why atRA therapy induces skin dryness when it is used topically and suggest that nicotinamide may be used as a moisturizer by down-regulating the expression of AQP3 in keratinocytes. Earlier studies have shown that topical nicotinamide improves skin barrier function, and nicotinamide cream is an effective moisturizer on atopic dry skin and may be used as an auxiliary medicine to treat atopic dermatitis. For example, the administration with myristyl nicotinate for one month has been found to reduce skin TEWL and provide additional barrier protection and tolerability of retinoic acid without interfering with improving efficacy.
Thus, AQPs appear to be key protein targets to improve the resistance and quality of the skin surface as well as to improve aging and sun exposure-induced dryness as shown by their roles in (1) hydrating the living layers of the epidermis where the keratinocyte differentiation takes place and (2) improving barrier formation, function and recovery. Indeed, considerable effort have been undertaken to develop techniques and compositions for improving skin appearance by stimulating aquaporins. For example, Breitenbach et. al. (US 2009/1030223; WO 2007/124991) describe a method of stimulating aquaporin expression in skin by contacting the skin with at least one of a glyceryl glycoside and a derivative thereof in an amount which is effective for stimulating aquaporin expression in the skin. Sene et. al. (US 2009/0036402) describe a composition for activating at least one of AQP-3, filaggrin or transglutaminase in the skin of an animal, comprising at least one compound from Centella Asiatica selected from the group consisting of madecassoside, terminoloside, asiaticoside, madecassic acid, asiatic acid and mixtures thereof. Xie et. al. (US 2007/0009474) describe personal care compositions comprising from about 0.05% to about 5% of at least one aquaporin-stimulating compound selected from the group consisting of xanthine, caffeine; 2-amino-6-methyl-mercaptopurine; 1-methyl xanthine; 2-aminopurine; theophylline; theobromine; adenine; adenosine; kinetin; p-chlorophenoxyacetic acid; 2,4-dichlorophenoxyacetic acid; indole-3-butyric acid; indole-3-acetic acid methyl ester; beta-naphthoxyacetic acid; 2,3,5-triiodobenzoic acid; adenine hemisulfate; n-benzyl-9-(2-tetrahydropyranyl)adenine; 1,3-diphenylurea; 1-phenyl-3-(1,2,3-thiadiazol-5-yl)urea; zeatin; indole-3-acetic acid; 6-benzylaminopurine; alpha-napthaleneacetic acid; 6-2-furoylaminopurine; green tea extract; white tea extract; menthol; tea tree oil; ginsenoside-RB1; ginsenoside-RB3; ginsenoside-RC; ginsenoside-RD; ginsenoside-RE; ginsenoside-RG1; ginseng root extract; ginseng flower extract; pomegranate extract, extracts from Ajuga turkestanica; extracts from viola tricolor and combinations thereof; an additional ingredient selected from the group consisting of niacinamide, glycerin and mixtures thereof, and a dermatologically-acceptable carrier. Thiem et. al. (EP0770378) describes cosmetic or pharmaceutical preparations comprising hexosylglycerides and/or (hexosyl)hexosylglycerides as well as the use of glycosyl glycerides as agents which enhance skin moistness. Such studies are not limited to the patent literature, as numerous technical articles have been presented on the subject as well including: M Dumas, C Gondran, P Barré, R Sougrat, J M Verbavatz, C Heusèle, S Schnébert, F Bonté, Effect of an Ajuga turkestanica extract on aquaporin 3 expression, water flux, differentiation and barrier parameters of the human epidermis, Eur J Dermatol, 12 (6):XXV-XXVI, 2002 and M Zelenina, S Tritto, A A Bondar, S Zelenin, A Aperia, Copper inhibits the water and glycerol permeability of aquaporin-3, J Biol Chem, 279 (50):51939-51943, 2004.
Another factor key to TEWL and the integrity of TJs are the zonula occludens. Zonula occluden (ZO) proteins, comprising ZO-1, -2, and -3, are peripheral proteins localizing at junctional sites. ZO proteins are scaffolding proteins recruiting various types of proteins to the cytoplasmic surface of the junction, thereby contributing to the so called “junctional plaque”. ZO proteins have originally been described to localize specifically to tight junctions (TJs) (zonulae occludentes) [B. R. Stevenson, J. D. Siliciano, and M. S. Mooseker, “Identification of ZO-1: a high molecular weight polypeptide associated with the tight junction (Zonula Occludens) in a variety of epithelia,” Journal of Cell Biology, vol. 103, no. 3, pp. 755-766, 1986.]. However, this notion was quickly reevaluated, since these proteins were found to associate with the cadherin-based adherens junctions (AJs) in cells lacking TJs [A. G. Howarth, M. R. Hughes, and B. R. Stevenson, “Detection of the tight junction-associated protein ZO-1 in astrocytes and other nonepithelial cell types,” American Journal of Physiology, vol. 262, no. 2, pp. C461-C469, 1992].
Moreover, ZO proteins also associate with gap junctions (GJs) by directly interacting with connexins [H. Bauer, J Zweimueller-Mayer, P. Steinbacher, A. Lametschwandtner, and H. C. Bauer, The Dual Role of Zonula Occludens (ZO) Proteins, J Biomed and Biotech, vol. 2010, Article ID 402593, 11 pages, 2010. doi:10.1155/2010/402593], which points towards a general role of ZO proteins in intercellular adhesion and communication. The most prominent function of ZO proteins at the junctional site is the regulation of claudin polymerization in epithelial cells, which was demonstrated by use of a reverse genetic approach [K. Umeda, J. Ikenouchi, S. Katahira-Tayama, et al., “ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation,” Cell, vol. 126, no. 4, pp. 741-754, 2006].
In recent years, intriguing evidence has accumulated suggesting that ZO proteins not only exert functions related to structural barrier mechanisms but are also involved in signal transduction and transcriptional modulation [H. Bauer, J Zweimueller-Mayer, P. Steinbacher, A. Lametschwandtner, and H. C. Bauer, The Dual Role of Zonula Occludens (ZO) Proteins, J Biomed and Biotech, vol. 2010, Article ID 402593, 11 pages, 2010. doi:10.1155/2010/402593].
Despite these findings and the advances made, there is still a definite need for skin care/treatment compositions that are more effective and more forgiving; especially those that are able to improve skin health and appearances. However, in contrast to early efforts, a more fundamental and comprehensive approach is needed for improving skin health and appearances that is based on the biology of the skin. As noted above, decline of skin health and appearance is a natural phenomenon that occurs over time and is not just a result of wear and tear, but is also the consequence of a continually active genetic program that might be up- or down-regulated resulting in detrimental effects on skin. Thus, from a biological standpoint, an effective strategy for improving the health and appearance of skin must include ingredient(s) that provide(s) hydration of the dermis and epidermis and that regulate(s) both epidermal differentiation and lipid synthesis/secretion, which in turn influence permeability barrier homeostasis. Retinoids, while effective, have poor stability and at high levels, especially on a continual basis, results in other adverse consequences including skin sensitization and irritation. Glycerol is effective, but it is tacky and requires high level use. Additionally, glycerol does not give aesthetically pleasing formulations due to tackiness. Glycerylglycosides are not very stable, therefore difficult to formulate and the formulated products have shorter shelf-life.
Plant extracts have found great utility in skin care products; however, their use is not a simple matter. Plant extracts are by nature very complex having numerous constituents in varying concentrations. Identification and selection of the proper plant is critical, even the type of plant, e.g. chemo-, pheno-, and geno-type, which will have a marked influence on the active ingredients. Similarly, the portion of the plant to be used in the extraction process is also important as there are marked differences in the nature and abundance of the chemical constituents in the roots, leaves, bark, and other parts of the plants. Harvesting of the plants also influences the chemical constituents and their concentrations as once harvested certain chemicals may degrade or become more sensitive to degradation by environmental factors. All of these need to be considered and accounted for in the use of plant extracts, particularly for ensuring that the extracts will have the appropriate concentrations and ratios necessary to elicit the desired skin care benefits. The key to getting consistent and predictable results is to have standardized plant extracts, which is seldom the case in commercially available material.
Thus, in light of the foregoing discussion, there continues to be a need for compounds that are capable of stimulating the aquaporin membrane proteins, especially ones that will increase the expression of AQP-3, so as to improve skin hydration and thereby minimize the visual signs of dry or photo-damaged skin, enhancing skin moisturization, appearance, tone, texture and firmness.
Additionally, there continues to be a need for compounds that are capable of stimulating key genes/proteins associated with the tight junctions, desmosomes, and epidermal differentiation for maintaining and/or improving barrier formation, function and recovery in mammalian skin.